SAMHD1 is a cellular protein playing key roles in the innate immune system, which inhibits infection of blood cells by a wide range of retroviruses including HIV-1 and certain DNA viruses. It also prevents the development of the autoinflammatory Aicardi-Goutires syndrome (AGS). SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that degrades the intracellular pool of deoxynucleoside triphosphates (dNTPs) during early reverse transcription. SAMHD1 is also a 3'-5' exonuclease that nonspecifically degrades single stranded (ss) RNA and ssDNA. Notably, phosphorylation of SAMHD1 at residue T592 in cycling cells impairs its ability to restrict HIV-1. Despite intensive investigation, the mechanism for viral restriction by SAMHD1 still remains unclear and controversial. The overall goal of the proposed research is to establish the biochemical and structural basis by which SAMHD1 inhibits retroviral infection and to delineate the cellular function of its nuclease activities. To achieve this goal, we will use a combination o techniques including biochemistry, biophysics, structural biology, and cell biology to delineate the catalytic mechanisms of the multiple enzymatic activities of SAMHD1 and further elucidate the relevance of each enzymatic activity in its antiviral and other cellular functions. We have established robust in vitro assays to obtain a rigorous measurement of the dNTPase activity and the nuclease activity of SAMHD1. Based on our crystal structure of the SAMHD1 tetramer, we have identified residues important only for the dNTPase activity. We will perform structural-guided mutagenesis to design mutants with decoupled dNTPase and nuclease activities. We will also identify the ssRNA/ssDNA binding sites and identities of the metal ions at the nuclease catalytic sites to facilitate the design of such mutants and to advance the mechanistic understanding of the multiple activities of SAMHD1. The mutants will be verified in our in vitro assays and used in cellular viral restriction assays to assess their relevance to the antiviral activity of SAMHD1. Concurrent with these studies, we will determine the crystal structures of SAMHD1 in complex with ssRNA and ssDNA and validate the structural observations. The detailed structural information on SAMHD1-nucleic acid interactions is essential for revealing the catalytic mechanism of SAMHD1 nuclease activity. We will also investigate the role of the nuclease activity of SAMHD1 in cells by examining the cellular interferon stimulation in response to the nucleic acids in the presence of SAMHD1 mutants. We will further establish the production of fully phosphorylated SAMHD1 in a specialized E. coli expression system to elucidate the impact of T592 phosphorylation on its activities by biochemical and structural studies. A complete understanding of the regulation and modulation of the enzymatic activities of SAMHD1 in HIV restriction is crucial for assessing its potential as a therapeutic target for AIDS.